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Biochemical and Structural Elucidation of Polyketide Synthase and Non-Ribosomal Peptide Synthetase Enzymes Using Novel Pantetheine Analogues


Polyketide derived natural products are a large and diverse class of secondary metabolites used in the pharmaceutical and agricultural industry. These include antibiotics, immunosuppressant's, chemotherapeutics and insecticides. Examples of valuable polyketide compounds include tetracycline (antibiotic), lovastatin (cholesterol-lowering) and rapamycin ( immunosuppressant). The multi-domain enzyme complex that is responsible for the biosynthesis of polyketides is known as the polyketide synthase (PKS). Similar to the PKS, the fatty acid synthase (FAS) is a multi-domain enzyme complex that utilizes acyl-CoA's as building blocks to generate linear poly-beta keto intermediates that can be subsequently processed via reduction, dehydration or other acyl-chain modifying reactions. Both FAS and PKS utilize the acyl carrier protein (ACP) that functions as the transporter protein that is covalently tethered to the nascent polyketide intermediate. Polyketide and fatty acid production require three basic biosynthetic steps: (1) polyketide elongation, (2) polyketide modification and (3) polyketide release from the ACP. How the ACP interacts with enzyme domains throughout these steps is not well understood. Unlike FAS, PKS utilize more diverse polyketide starter units and undergo various modification reactions that give rise to the rich chemical diversity of polyketide natural products. One of these important modifications includes the regio-specific cyclization and aromatization of linear poly-β-keto intermediates. How the different PKS promote different cyclization patterns is not well understood. Lastly, how polyketide ACP-tethered intermediates are released from the PKS machinery needs further investigation. An elucidation of the mechanism by which these bioactive polyketides are cyclized and released while being tethered to the ACP would be highly significant in the prediction, identification and engineered biosynthesis of new polyketides.

The objective of this dissertation is to better understand the mechanism of polyketide cyclization, product release and elucidate on the structural role ACP plays in recognizing various modification enzymes. These objectives will be further divided into three aims.

Aim 1: Understand polyketide substrate specificity to further elucidate on the mechanism of regio-specific polyketide cyclization. We co-crystallized novel pantetheine tethered linear polyketide probes to precisely define the binding motif of a polyketide intermediate in the active site of the aflatoxin product template domain (PksA PT). A systematic mutational survey of the PksA PT was utilized for confirmation.

Aim 2: Identify important protein-protein interactions between ACPs and other PKS enzymes. By utilizing mechanism based protein-protein crossliking and in vitro reconstitution assays we identified key residues responsible for surface interaction between ACPs and PT domains. Site-directed mutagenesis confirmed the important of key surface residues.

Aim 3: Elucidate the mechanism of a type I modular polyketide synthase/ non-ribosomal peptide synthetase (PKS-NRPS) releasing reductase (R) domain. We solved the crystal structure of the R domain from the myxalamid biosynthetic pathway and conducted a series of systematic mutations to identify key residues important in electron transfer and substrate specificity. Using molecular dynamics, we identified flexible regions responsible for substrate and co-factor binding.

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